MaxPeak High Performance Surfaces Enable Reproducibility and Enhanced Experimental Robustness in High Throughput Proteomics

Robust and reproducible LC-MS is an essential requirement in large scale clinical proteomic experiments, and the available sample amounts lend themselves to the use of larger I.D. column geometries compared to traditional proteomic experiments.

A quality control sample of a complex tryptic digest was injected nearly two hundred times onto a MaxPeak High Performance Surfaces Technology enabled LC system, the ACQUITY PREMIER, fitted with a CSH 2.1 mm I.D. column and eluent coupled to a SYNAPT XS Mass Spectrometer. The injections of an E. coli digest onto the CSH column took place at set intervals inside a larger clinical proteomic study. The raw and processed data is used to showcase excellent reproducibility and robustness of several characteristics of the liquid chromatograph (LC) and mass spectrometer (MS) used in the experiment – information which is important for both the QC of the study and applicability to wider clinical proteomic cohorts.

Chromatographic parameters; peak width and retention time reproducibility, are assessed across all the injections with over 800 protein and over 1 million individual peptide measurements contributing. The consistency of measured peptide intensities and protein sequence coverage leads to a large percentage of total proteins being identified across an experiment which spanned over a six week period.

Benefits

• HPS technology removes any issues with metal adsorbed species
• Routine, robust, and reproducible analytical scale LC-MS
• Suitable for large cohort, clinical proteomic analyses

Increased interest in the field of clinical proteomics, where sample amounts are not limited, has led to various studies into the utilization of larger scale chromatography than would typically be used for more traditional, nanoflow-based proteomic experiments. Larger scale chromatography tends to be more user friendly, robust, and reproducible than the traditional nanoscale proteomics set up¹ which can be sometimes seen as only accessible to an expert user.

Waters developed a class of new technologies, known as MaxPeak High Performance Surfaces (HPS), and these have been applied in the development of a new liquid chromatographic system called the ACQUITY PREMIER.² While primarily aimed at limiting adsorption of certain compounds to metal surfaces and thus enhancing their detection, investigations suggest that a user may be completely unaware they are losing information from any sample being analyzed. This is particularly concerning in untargeted, discovery type analyses and it therefore follows that the use of MaxPeak HPS Technology is preferable for any experimental procedure.

A large-scale plasma proteomic experiment of over 500 samples, injected three times each, was carried out and during this analysis, after every 8 samples, a quality control injection of E. coli tryptic digest was performed. By analyzing these QC results, it is possible to derive metrics for the reproducibility and robustness of the entire experiment. This application brief aims to show that over the six-week duration of the study, outstanding reproducibility and robustness, both chromatographic and mass spectrometric, is observed.

Sample Description

A sample of 2 μg MPDS E. coli (186003196), was injected every 4 hours in-between injections of plasma tryptic digests that were being analyzed for the wider scale clinical proteomics experiment. Throughout the six week experiment, the E. coli sample was injected over 180 times and processed using Progenesis QIP, PLGS, and data viewed with Spotfire data visualization software.

Reproducibility – Chromatography

Figure 1A shows the raw data chromatograms from two injections (30 days apart), with Figure 1B representing a retention time window from 5 to 8.5 minutes. Figure 2, the distribution of chromatographic peak widths of all identified peptides, shows the majority eluting over 3 seconds FWHM. Further to this, Figure 3 represents the frequencies of retention time measurements for a selection of precursor ions from all the injections associated with tryptophanase (P0A853; TNAA_ECOLI). Coefficient of variation (CV) of 1% or less are observed, showing clearly that excellent chromatographic retention time reproducibility has been maintained across the experiment.

LC-MS Robustness – Peptide Intensities and Mass Accuracy

One of the outputs generated from PQIP and PLGS processing for identified proteins and peptides is the sum of product ion intensities for each peptide and can be used as a means of monitoring intensity robustness. The results for three E. coli related proteins are shown in Figure 4, with ions from the most intense protein tryptophanase (TNAA_ECOLI) and two less intense proteins, malate dehydrogenase (P61889; MDH_ECOLI), and aldehyde-alcohol dehydrogenase (P0A9Q7;ADHE_ECOLI), shown. Further evidence of the robustness of the experiment is given by the peptide precursor mass accuracy, where it is found that >95% of peptides are measured within ±5 ppm of their theoretical value, Figure 5.

MS Robustness – Protein Sequence Coverage and Identifications

One parameter derived is the protein sequence coverage resulting from peptide identifications for each protein. For one protein TNAA_ECOLI, an average sequence coverage of 76% (CV 2.8%) is observed across the experiment, Figure 6A. Figure 6B is a distribution graph showing the ranges of the sequence coverage for all identified proteins and shows a modal range of 40 to 49%. The goal of many proteomic experiments is to first identify proteins prior to determining any regulation of these when comparing different samples/conditions. However, for these QC analyses where the same sample has been injected, the reproducibility of identifications throughout the five-week duration has been measured. Figure 7 shows a Venn diagram whereby identifications comprised of three injections, which correspond to the early (5 days), middle (18 days), and late (38 days) stages of the experiment are combined and compared. This suggests that approximately 76% of proteins are identified in injections across the whole experiment.

MaxPeak HPS Technology ensures that adsorption to column hardware material is minimized. The routine nature of analytical scale chromatography suggests an experimental avenue when sample limitations are not an issue, allowing for high throughput analyses. We have shown results from analyzing a QC sample injected within a large scale proteomic experiment, utilizing an LC system and column that have been treated with MaxPeak High Performance Technology, and have shown this LC-MS configuration with SYNAPT XS to exhibit excellent robustness and reproducibility. Over the course of a six week LC-MS study, data metrics derived from the interspersed QC’s demonstrate excellent reproducibility of peptide intensities and precursor retention times. Resulting protein identifications and their sequence coverage is also shown to be maintained throughout the study.

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